Human pelota homolog

ABSTRACT

68772 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing 68772 polypeptides and polynucleotides in the design of protocols for the treatment of proliferative diseases such as leukemias, solid tumor cancers and metastases; chronic inflammatory proliferative diseases such as psoriasis and rheumatoid arthritis; proliferative cardiovascular diseases such as restenosis; prolifertive ocular disorders such as diabetic retinopathy; and benign hyperproliferative diseases such as hemangiomas, among others, and diagnostic assays for such conditions.

FIELD OF INVENTION

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by them and to the use of such polynucleotides andpolypeptides, and to their production. More particularly, thepolynucleotides and polypeptides of the present invention relate toPelota family, hereinafter referred to as 68772. The invention alsorelates to inhibiting or activating the action of such polynucleotidesand polypeptides.

BACKGROUND OF THE INVENTION

[0002] Regulation of the cell cycle is controlled by a family ofcyclins, cyclin dependent kinases (CDKs), CDK regulatory kinases, andphosphatases. See, Lees, E., Curr. Opin. Cell. Biol. 1995, 7:773-780;Piwinica-Worms, H., J. Lab. Clin. Med. 1996, 128:350-354. Progressionfrom the G2 phase to the M phase of the cell cycle requires theactivityof cdc25 phosphatase. In Drosophila, mutations in the pelotagene have the same phenotype as mutations in the twine and/or sag genes,which are cdc25 homologs, Eberhart, C. G. and Wasserman. S. A., Devel.1995, 121:3477-3486. Specific cell cycle effects of pelota mutations areseen in both meiosis and mitosis, including G2/M arrest between mitoticand meiotic cell division, and disruption of nuclear envelope breakdownand spindle formation. Regulation of pelota offers a means ofcontrolling a critical event in the cell cycle.

[0003] This indicates that the Pelota family has an established, provenhistory as therapeutic targets. Clearly there is a need foridentification and characterization of further members of Pelota familywhich can play a role in preventing, ameliorating or correctingdysfunctions or diseases, including, but not limited to, proliferativediseases such as leukemias, solid tumor cancers and metastases; chronicinflammatory proliferative diseases such as psoriasis and rheumatoidarthritis; proliferative cardiovascular diseases such as restenosis;prolifertive ocular disorders such as diabetic retinopathy; and benignhyperproliferative diseases such as hemangiomas.

SUMMARY OF THE INVENTION

[0004] In one aspect, the invention relates to 68772 polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such 68772 polypeptidesand polynucleotides. Such uses include the treatment of proliferativediseases such as leukemias, solid tumor cancers and metatases; chronicinflammatory proliferative diseases such as psoriasis and rheumatoidarthritis; proliferative cardiovascular diseases such as restenosis;prolifertive ocular disorders such as diabetic retinopathy; and benignhyperproliferative diseases such as hemangiomas, among others. In stillanother aspect, the invention relates to methods to identify agonistsand antagonists using the materials provided by the invention, andtreating conditions associated with 68772 imbalance with the identifiedcompounds. Yet another aspect of the invention relates to diagnosticassays for detecting diseases associated with inappropriate 68772activity or levels.

DESCRIPTION OF THE INVENTION

[0005] Definitions

[0006] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0007] “68772” refers, among others, generally to a polypeptide havingthe amino acid sequence set forth in SEQ ID NO:2 or an allelic variantthereof.

[0008] “68772 activity or 68772 polypeptide activity” or “biologicalactivity of the 68772 or 68772 polypeptide” refers to the metabolic orphysiologic function of said 68772 including similar activities orimproved activities or these activities with decreased undesirableside-effects. Also included are antigenic and immunogenic activities ofsaid 68772.

[0009] “68772 gene” refers to a polynucleotide having the nucleotidesequence set forth in SEQ ID NO: 1 or allelic variants thereof and/ortheir complements.

[0010] “Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0011] “Isolated” means altered “by the hand of man” from the naturalstate. If an “isolated” composition or substance occurs in nature, ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livinganimal is not “isolated,” but the same polynucleotide or polypeptideseparated from the coexisting materials of its natural state is“isolated”, as the term is employed herein.

[0012] “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single-and double-strandedregions, single- and double-stranded RNA, and RNA that is mixture ofsingle- and double-stranded regions, hybrid molecules comprising DNA andRNA that may be single-stranded or, more typically, double-stranded or amixture of single- and double-stranded regions. In addition,“polynucleotide” refers to triple-stranded regions comprising RNA or DNAor both RNA and DNA. The term polynucleotide also includes DNAs or RNAscontaining one or more modified bases and DNAs or RNAs with backbonesmodified for stability or for other reasons. “Modified” bases include,for example, tritylated bases and unusual bases such as inosine. Avariety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0013] “Polypeptide” refers to any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres. “Polypeptide” refers to bothshort chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-liking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Acadermic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

[0014] “Variant” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

[0015] “Identity” is a measure of the identity of nucleotide sequencesor amino acid sequences. In general, the sequences are aligned so thatthe highest order match is obtained. “Identity” per se has anart-recognized meaning and can be calculated using published techniques.See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOMEPROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTERANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey; 1994; SEQUENCE ANALYSIS IN MOLECULARBIOLOGY, von Heinje, G., Academnic Press, 1987; and SEQUENCE ANALYSISPRIMIER, Gribskov, M. and Devereux, J., eds., M Stockton Press, NewYork, 1991). While there exist a number of methods to measure identitvbetween two polynucleotide or polypeptide sequences, the term “identity”is well known to skilled artisans (Carillo, H., and Lipton, D., SLAM JApplied Math (1988) 48:1073). Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to, those disclosed in Guide to Huge Computers, Martin J.Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., andLipton, D., SLAM J Applied Math (1988) 48:1073. Methods to determineidentity and similarity are codified in computer programs. Preferredcomputer program methods to determine identity and sirilaritv betweentwo sequences include, but are not limited to, GCS program package(Devereux, J., er al., Nucleic Acids Research (1984) 12(1):387), BLASTP,BLASTN, FASTA (Atschul, S. F. et al., J Molec Biol (1990) 215:403).

[0016] As an illustration, by a polynucleotide having a nucleotidesequence having at least, for example, 95% “identity” to a referencenucleotide sequence of SEQ ID NO: 1 is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations per each 100 nucleotides of the reference nucleotide sequenceof SEQ ID NO: 1. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0017] Similarly, by a polypeptide having an amino acid sequence havingat least, for example, 95% “identity” to a reference amino acid sequenceof SEQ ID NO:2 is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0018] Polypeptides of the Invention

[0019] In one aspect, the present invention relates to 68772polypeptides (or 68772 proteins). The 68772 polypeptides include thepolypeptide of SEQ ID NO:2; as well as polypeptides comprising the aminoacid sequence of SEQ ID NO: 2; and polypeptides comprising the aminoacid sequence which have at least 80% identity to that of SEQ ID NO:2over its entire length, and still more preferably at least 90% identity,and even still more preferably at least 95% identity to SEQ ID NO: 2.Furthermore, those with at least 97-99% are highly preferred. Alsoincluded within 68772 polypeptides are polypeptides having the aminoacid sequence which have at least 80% identity to the polypeptide havingthe amino acid sequence of SEQ ID NO:2 over its entire length, and stillmore preferably at least 90% identity, and still more preferably atleast 95% identity to SEQ ID NO:2. Furthermore, those with at least97-99% are highly preferred. Preferably 68772 polypeptide exhibit atleast one biological activity of 68772.

[0020] The 68772 polypeptides may be in the form of the “mature” proteinor may be a part of a larger protein such as a fusion protein. It isoften advantageous to include an additional amino acid sequence whichcontains secretory or leader sequences, pro-sequences, sequences whichaid in purification such as multiple histidine residues, or anadditional sequence for stability during recombinant production.

[0021] Fragments of the 68772 polypeptides are also included in theinvention. A fragment is a polypeptide having an amino acid sequencethat entirely is the same as part, but not all, of the amino acidsequence of the aforementioned 68772 polypeptides. As with 68772polypeptides, fragments may be “free-standing,” or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region. Representative examples of polypeptidefragments of the invention, include, for example, fragments from aboutamino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the endof 68772 polypeptide. In this context “about” includes the particularlyrecited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acidat either extreme or at both extremes.

[0022] Preferred fragments include, for example, truncation polypeptideshaving the amino acid sequence of 68772 polypeptides, except fordeletion of a continuous series of residues that includes the aminoterminus, or a continuous series of residues that includes the carboxylterminus or deletion of two continuous series of residues, one includingthe amino terminus and one including the carboxyl terminus. Alsopreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophobicregions, hydrohobic regons, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions, substate bindingregion, and high antigenic index regions. Other preferred fragments arebiologically active fragments. Biologically active fragments are thosethat mediate 68772 activity, including those with a similar activity oran improved activity, or with a decreased undesirable activity. Alsoincluded are those that are antigenic or immunogenic in an animalespecially in a human.

[0023] Preferably, all of thee polypeptide fragments retain thebiological activity of the 68772, including antigenic activity. Variantsof the defined sequence and fragments also form part of the presentinvention. Preferred variants are those that vary from the referents byconservative amino acid substitutions-i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and lie; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several 5-10, 1-5, or 1-2 amino acids aresubstituted deleted, or added in any combination.

[0024] The 68772 polypeptides of the invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0025] Polynucleotides of the Invention

[0026] Another aspect of the invention relates to 68772 polynucleotides.68772 polynucleotides include isolated polynucleotides which encode the68772 polypeptides and fragments, and polynucleotides closely relatedthereto. More specifically, 68772 polynucleotide of the inventioninclude a polynucleotide comprising the nucleotide sequence set forth inSEQ ID NO:1 encoding a68772 polypepide of SEQ ID NO: 2, andpolynucleotide having the particular sequence of SEQ ID NO: 1. 68772polynucleotides further include a polynucleotide comprising a nucleotidesequence that has at least 80% identity over its entire length to anucleotide sequence encoding the 68772 polypeptide of SEQ ID NO:2, and apolynucleotide comprising a nucleotide sequence that is at least 80%identical to that of SEQ ID NO: 1 over its entire length. In thisregard, polynucleotides at least 90% identical are particularlypreferred, and those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred and those withat least 98-99% are most highly preferred, with at least 99% being themost preferred. Also included under 68772 polynucleotides are anucleotide sequence which has sufficient identity to a nucleotidesequence contained in SEQ ID NO: 1 or contained in the cDNA insert inthe plasmid deposited with the ATCC Deposit number ATCC 98438 tohybridize under conditions useable for amplification or for use as aprobe or marker. Moreover, 68772 polynucleotide include a nucleotidesequence having at least 80% identity to a nucleotide sequence encodingthe 68772 polypeptide expressed by the cDNA insert deposited at the ATCCwith Deposit Number ATCC 98438, and a nucleotide sequence comprising atleast 15 contiguous nucleotides of such cDNA insert. In this regard,polynucleotides at least 90% identical are particularly preferred, andthose with at least 95% are especially preferred. Furthermore, thosewith at least 97% are highly preferred and those with at least 98-99%are most highly preferred, with at least 99% being the most preferred.The invention also provides polynucleotides which are complementary toall the above 68772 polynucleotides.

[0027] A deposit containing a human 68772 cDNA has been deposited withthe American Type Culture Collection (ATCC), 12301 Park Lawn Drive,Rockville, Md. 20852, U.S.A., on May 28, 1997, and assigned ATCC DepositNumber ATCC 98438. The deposited material (clone) is SOLR containingUniZap (Stratagene, La Jolla, Calif. ) that further contains the fulllength 68772 cDNA, referred to as “Human pelota cDNA clone from a humanT-cell library, ATG-1030” upon deposit. The cDNA insert is within EcoRI, Xho I site(s) in the vector. The nucleotide sequence of thepolynucleotides contained in the deposited material, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

[0028] The deposit has been made under the terms of the Budapest Treatyon the international recognition of the deposit of micro-organisms forpurposes of patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §12.

[0029] 68772 of the invention is structurally related to other proteinsof the Pelota family, as shown by the results of sequencing the cDNA ofTable 1 (SEQ ID NO: 1) encoding human 68772. The cDNA sequence of SEQ IDNO: 1 contains an open reading frame (nucleotide number 250 to 1407)encoding a polypeptide of 385 amino acids of SEQ ID NO:2. Amino acidsequence of Table 2 (SEQ ID NO:2) has about 65% identity (using Gap inGCG (Needleman Wunsch)) in 395 amino acid residues with Drosophilamelanogaster pelota (Eberhart and Wasserman, Devel. 121:3477-3486,1995). Furthermore, 68772 (SEQ ID NO:2) is 36% identical toSaccharomyces cerevisiae DOM34 over 387 amino acid residues (Lalo etal., Compets Rendus de l'Academie des Sciences 316:367-373, 1993).Nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 63% identity(using Gap in GCG (Nedm Wunsch)) in 1186 nucleotide residues withDrosophila melanogaster pelota (Eberhart and Wasseman, Devel.121:3477-3486, 1995). Furthermore, 68772 (SEQ ID NO: 1) is 45% identicalto Saccharomyces cerevisiae DOM34 over 1176 nucleotide base residues(Lalo et al., Compets Rendus de l'Acadermie des Sciences 316:367-373,1993) Thus 68772 polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides, and their utility is obvious to anyone skilled in theart. TABLE 1^(a)CCCGGGCGCTGCAGTGTTCCCCGAGCOTGTTAGACGCAGCGCGCCGGGAGACTGAGAGAGGAAAGGATAGAGGAAGTGCTGCCCTAGGCTGCATGAGTCGAAGCAAGCGTGTTTCCTTCCCGCCAGGCAAGTGCCCTTAGAAACCGGGCCCCGCCCCCTTCCTGGCCTGCATTCCCATCCCCTCTCCCGGGGCGGAGGTGAGGACCTCCTTGGTTCCTTTGGTTCTGTCAGTGAGCCCCTTCCTTGGCCATGAAGCTCGTGAGGAAGAACATCGAGAAGGACAATGCGGGCCAGGTGACCCTGGTCCCCGAGGAGCCTGAGGACATGTGGCACACTTACAACCTCGTGCAGGTGGGCGACAGCCTGCGCGCCTCCACCATCCGCAAGGTACAGACAGAGTCCTCCACGGGCAGCGTGGGCAGCAACCGGGTCCGCACTACCCTCACTCTCTGCGTGGAGGCCATCGACTTCGACTCTCAAGCCTGCCAGCTGCGGGTTAAGGGGACCAACATCCAAGACAATGAGTATGTCAAGATGGGGGCTTACCACACCATCGAGCTGGAGCCCAACCGCCAGTTCACCCTGGCCAAGAAGCAGTGGGATAGTGTGGTACTGGAGCGCATCGAGCAGGCCTGTGACCCAGCCTGGAGCGCTGATGTGGCGGCTGTCGTCATGCAGGAAGGCCTCGCCCATATCTGCTTAGTCACTCCCAGCATGACCCTCACTCGGGCCAAGGTGGACGTGAACATCCCTAGGAAAAGGAAAGGCAATTGCTCTCAGCATGACCGGCCCTTGGAGCGGTTCTATGAACAGGTGGTCCAGGCTATCCAGCGCCACATACACTTTGATGTTGTAAACTCCATCCTCGTGGCCAGCCCAGGATTTGTGAGGGAGCAGTTCTGCGACTACATGTTTCAACAAGCAGTGAAGACCGACAACAAACTGCTCCTGGAAAACCGGTCCAAATTTCTTCAGGTACATGCCTCCTCCGGACACAAGTACTCCCTGAAAGAGGCCCTTTGTGACCCTACTGTGGCTAGCCGCCTTTCAGACACTAAAGCTGCTGCGGPAGTCAPAGCCTTGGATGACTTCTATAAAATGTTACAGCATGAACCGGATCGAGCTTTCTATGGACTCAAGCAGGTGGAGAAGGCCAATGAAGCCATGGCAATTGACACATTGCTCATCAGCGATGAGCTCTTCAGGCATCAGGATGTAGCCACACGGAGCCGGTATGTGAGGCTGGTGGACAGTGTGAAAGAGAATGCAGGCACCGCTAGGATATTCTCTAGTCTTCACGTTTCTGGGGAACAGCTCAGCCJGTTGACTGGGGTAGCTGCCATTCTCCGCTTCCCTGTTCCCGAACTTTCTGACCAAGAGGGTGATTCCAGTTCTGAAGAGCATTAATGATTGAAACTTAAAATTGAGACAATCTTGTGTTTCCTAAACTGTTACAGTACATTTCTCAGCATCCTTGTGACAGAAAGCTGCAAGAAGGGCACTTTTTGATTCATACAGGGATTTCTTATGTCTTTGGCTACACTAGATATTTTGTGATTGGCAAGACATGTATTTAAACAATAAACTAAAAGGAAATAATCTCCACGTACTACCAAAAAAAAAAAAAAAAAA.

[0030] TABLE 2^(b)MKLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTESSTGSVGSNRVRTTLTLCVEAIDFDSQACQLRVKGTNIQENEYVKMGAYHTIELEPNRQFTLAKKQWDSVVLERIEQACDRAWSADVAAVVMQEGLAHICLVTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDVVKCILVASPGFVREQFCDYMFQQAVKTDNKLLLENRSKFLQVHASSGHKYSLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRAFYGLKQVEKANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTARIFSSLHVSGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED.

[0031] One polynucleotide of the present invention encoding 68772 may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells of human activated T-Cells, pancreas tumor,colon carcinoma, placenta, chondrosarcoma, hypoxic synoviocytes,osteoclastoma, tonsils, promyelocyte, heart, stimulated endothelialcells, and breast lymph node cells using the expressed sequence tag(EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams,M. D. et al., Nature, (1992)355:632-634; Adams, M. D., et al., Nature(1995)377 Supp:3-174). Polynucleotides of the invention can also beobtained from natural sources such as genomic DNA libraries or can besynthesized using well known and commercially available techniques.

[0032] The nucleotide sequence encoding 68772 polypeptide of SEQ ID NO:2may be identical to the polypeptide encoding sequence contained in Table1 (nucleotide number 250 to 1407 of SEQ ID NO: 1), or it may be asequence, which as a result of the redundancy (degeneracy) of thegenetic code, also encodes the polypeptide of SEQ ID NO:2.

[0033] When the polynucleotides of the invention are used for therecombinant production of 68772 polypeptide, the polynucleotide mayinclude the coding sequence for the mature polypeptide or a fragmentthereof, by itself, the coding sequence for the mature polypeptide orfragment in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro-orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence which facilitates purification of the fusedpolypeptide can be encoded. In certain preferred embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al, Proc Natl Acad Sci U.S.A. (1989) 86:821-824, or is an HAtag. The polynucleotide may also contain noncoding 5′ and 3′ sequences,such as transcribed, non-translated sequences, splicing andpolyadenylation signals, ribosome binding sites and sequences thatstabilize mRNA.

[0034] Further preferred embodiments are polynucleotides encoding 68772variants comprise the amino acid sequence 68772 polypeptide of Table 2(SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acidresidues are substituted, deleted or added, in any combination.

[0035] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 80%, and preferablyat least 90%, and more preferably at least 95%, yet even more preferably97-99% identity between the sequences.

[0036] Polynucleotides of the invention, which are identical orsufficiently identical to a nucleotide sequence contained in SEQ ID NO:1or a ragment thereof, or to the cDNA insert in the plasmid deposited atthe ATCC with Deposit Number ATCC 98438 or a fragment thereof, may beused as hybridization probes for cDNA and genomic DNA, to isolatefull-length cDNAs and genomic clones encoding 68772 polypeptide and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the 68772 gene. Such hybridization techniques are known tothose of skill in the art. Typically these nuclectide sequences are 80%identical, preferably 90% identical, more preferably 95% identical tothat of the referent. The probes generally will comprise at least 15nucleotides. Preferably, such probes will have at least 30 nucleotidesand may have at least 50 nucleotides. Particularly preferred probes willrange between 30 and 50 nucleotides.

[0037] In one embodiment, to obtain a polynucleotide encoding 68772polypeptide comprises the steps of screening an appropriate libraryunder stingent hybridization conditions with a labeled probe having theSEQ ID NO: 1 or a fragment thereof, and isolating full-length cDNA andgenomic clones containing said polynucleotide sequence. Thus in anotheraspect, 68772 polynucleotides of the present invention further include anucleotide sequence comprising a nucleotide sequence that hybridizeunder stringent condition to a nucleotide sequence having SEQ ID NO: 1or a fragment thereof Also included with 68772 polypeptides arepolypeptide comprising antino acid sequence encoded by nucleotidesequence obtained by the above hybridization condition. Suchhybridization techniques are well known to those of skill in the art.Stringent hybridization conditions are as defined above or,alternatively, conditions under overnight incubation at 42° C. in asolution comprising: 50% formamide, 5×SSC (150mM NaCl, 15mM trisodiumcitrate), 50 mM sodium phosphate pH7.6), 5×Denhardt's solution, 10%detran sulfate, and 0.20 microgram/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 656° C.

[0038] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to animal and human disease.

[0039] Vectors, Host Cells, Expression

[0040] The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

[0041] For recombinant production host cells can be geneticallyengineered to incorporate expression systems or portions thereof forpolynucleotides of the present invention. Introduction ofpolynucleotides into host cells can be effected by methods described inmany standard laboratory manuals, such as Davis et al, BASIC METHODS INMOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) such as calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection.

[0042] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, Streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 andBowes melanoma cells; and plant cells.

[0043] A great variety of expression Systems can be used. Such Systemsinclude, among others, chromosomal, episomal and virus-derived systems,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

[0044] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0045] If the 68772 polypeptide is to be expressed for use in screeningassays, generally, it is preferred that the polypeptide be produced atthe surface of the cell. In this event, the cells may be harvested priorto use in the screening assay. If 68772 polypeptide is secreted into themedium, the medium can be recovered in order to recover and purify thepolypeptide; if produced intracellularly, the cells must first be lysedbefore the polypeptide is recovered. 68772 polypeptides can be recoveredand purified from recombinant cell cultures by well-known methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingproteins may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

[0046] Diagnostic Assays

[0047] This invention also relates to the use of 68772 polynucleotidesfor use as diagnostic reagents. Detection of a mutated form of 68772gene associated with a dysfunction will provide a diagnostic tool thatcan add to or define a diagnosis of a disease or susceptibility to adisease which results from under-expression, over-expression or alteredexpression of 68772. Individuals carrying mutations in the 68772 genemay be detected at the DNA level by a variety of techniques.

[0048] Nucleic acids for diagnosis may be obtained from a subjectscells, such as from blood, urine, saliva, tissue biopsy or autopsymaterial. The genormic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other ampliication techniquesprior to analysis. RNA or cDNA may also be used in similar fashion.Deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to labeled 68772nucleotide sequences. Perfectly matched sequences can be distinguishedfrom mismatched duplexes by RNase digestion or by differences in meltingtemperatures. DNA sequence differences may also be detected byalterations in electrophoretic mobility of DNA fragments in gels, withor without denaturing agents, or by direct DNA sequencing. See, e.g.,Myers et al., Science (1985) 230:1242. Sequence changes at specificlocations may also be revealed by nuclease protection assays, such asRNase and S1 protection or the chemical cleavage method. See Cotton etal, Proc Natl Acad Sci U.S.A. (1985) 85: 4397-4401. In anotherembodiment, an array of oligonuclecodes probes comprising 68772nucleotide sequence or fragments thereof can be constructed to conductefficient screening of e g, genetic mutations. Array technology methodsare well known and have general applicability and can be used to addressa variety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability. (See for example: M. Chee etal., Science, Vol 274, pp 610-613 (1996)).

[0049] The diagnostic assays offer a process for diagnosing ordetermining a susceptibility to proliferative diseases such asleukemias, solid tumor cancers and metastases; chronic inflammatoryproliferative diseases such as psoriasis and rheumatoid arthritis;proliferative cardiovascular diseases such as restenosis; prolifertiveocular disorders such as diabetic retinopathy; and benignhyperproliferative diseases such as hemangiomas through detection ofmutation in the 68772 gene by the methods described.

[0050] In addition, proliferative diseases such as leukemias, solidtumor cancers and metastases; chronic inflammatory proliferativediseases such as psoriasis and rheumatoid arthritis; proliferativecardiovascular diseases such as restenosis; prolifertive oculardisorders such as diabetic retinopathy; and benign hyperproliferativediseases such as hemangiomas, can be diagnosed by methods comprisingdetermining from a sample derived from a subject an abnormally decreasedor increased level of 68772 polypeptide or 68772 mRNA. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods. Assay techniques that can be used todetermine levels of a protein, such as an 68772 polypeptide, in a samplederived from a host are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis and ELISA assays.

[0051] Thus in another aspect, the present invention relates to adiagonostic kit for a disease or suspectability to a disease,particularly proliferative diseases such as leukemias, solid tumorcancers and metastases; chronic imflammatory proliferative diseases suchas psoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertve ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, which comprises:

[0052] (a) a 68772 polynucleotide, preferably the nucleotide sequence ofSEQ ID NO: 1, or a fragment thereof;

[0053] (b) a nucleotide sequence complementary to that of (a);

[0054] (c) a 68772 polypeptide, preferably the polypeptide of SEQ ID NO:2, or a fragment thereof; or

[0055] (d) an antibody to a 68772 polypeptide, preferably to thepolypeptide of SEQ ID NO: 2.

[0056] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0057] Chromosome Assays

[0058] The nucleotide sequences of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. The mapping of relevant sequences tochromosomes according to the present invention is an important firststep in correlating those sequences with gene associated disease. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

[0059] The differences in the cDNA or genomic sequence between affectedand unaffected individuals can also be determined. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

[0060] Antibodies

[0061] The polypeptides of the invention or their fragments or analogsthereof or cells expressing them can also be used as immunogens toproduce antibodies immunospecific for the 68772 polypeptides. The term“immunospecific” means that the antibodies have substantial greateraffinity for the polypeptides of the invention that their affinity forother related polypeptides in the prior art.

[0062] Antibodies generated against the 68772 polypeptides can beobtained by administering the polypeptides or epitope-bearing fragments,analogs or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and theEBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCERTHERAPY, pp. ⁷⁷-96, Alan R. Liss, Inc., 1985).

[0063] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can also be adapted to produce single chainantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms including other mammals, may be used to expresshumanized antibodies.

[0064] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or to purify the polypeptidesby affinity chromatography.

[0065] Antibodies against 68772 polypeptides may also be employed totreat proliferative diseases such as leukemias, solid tumor cancers andmetastases; chronic inflammatory proliferative diseases such aspsoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, among others.

[0066] Vaccines

[0067] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with 68772 polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom proliferative diseases such as leukemias, solid tumor cancers andmetastases; chronic inflammatory proliferative diseases such aspsoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashenangiomas, among others. Yet another aspect of the invention relatesto a method of inducing immunological response in a mammal whichcomprises, delivering 68772 polypeptide via a vector directingexpression of 68772 polynucleotide in vivo in order to induce such animmunological response to produce antibody to protect said animal fromdiseases.

[0068] Further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a 68772 polypeptide wherein the composition comprises a 68772polypeptide or 68772 gene. The vaccine formulation may further comprisea suitable carrier. Since 68772 polypeptide may be broken down in thestomach, it is preferably administered parenterally (includingsubcutaneous, intramuscular, intravenous, intradermal etc. injection).Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation instonicwith the blood of the recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents or thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example, sealed ampoules and vials and may be stored in afreeze-dried condition requiring only the addition of the sterile liquidcarrier immediately prior to use. The vaccine formulation may alsoinclude adjuvant systems for enhancing the immunogenicity of theformulation, such as oil-in water systems and other systems known in theart. The dosage will depend on the specific activity of the vaccine andcan be readily determined by routine experimentation.

[0069] Screening Assays

[0070] The 68772 polypeptide of the present invention may be employed ina screening process for compounds which activate (agonists) or inhibitactivation of (antagonists, or otherwise called inhibitors) the 68772polypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess identify agonist or antagonistsfrom, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. These agonists or antagonists may benatural or modified substrates, ligands, receptors, enzymers, etc., asthe case may be, of the polypeptide of the present invention; or may bestructural or functional mimetic of the polypeptide of the presentinvention. See Coligan et al, Current Protocols in Immunology1(2):Chapter 5 (1991).

[0071] 68772 polypeptides are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate 68772 polypeptide on the one handand which can inhibit the function of 68772 polypeptide on the otherhand. I general, agonists are employed for therapeutic and prophylacticpurposes for such conditions as proliferative diseases such asleukemias, solid tumor cancers and metastases; chronic inflammatoryproliferative diseases such as psoriasis and rheumatoid arthritis;proliferative cardiovascular diseases such as restenosis; prolifertiveocular disorders such as diabetic retinopathy; and benignhyperproliferative diseases such as hemangiomas. Antagonists may beemployed for a variety of therapeutic and prophylactic purposes for suchconditions as proliferative diseases such as leukemias, solid tumorcancers and metastases; chronic inflammatory proliferative diseases suchas psoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas.

[0072] In general, such screening procedures may involve usingappropriate cells which express the 68772 polypeptide or respond to68772 polypeptide of the present invention. Such cells include cellsfrom mammals, yeast, Drosophila or. E. coli. Cells which express the68772 polypeptide (or cell membrane containing the expressedpolypeptide) or respond to 68772 polypeptide are then contacted with atest compound to observe binding or stimulation or inhibition of afunctional response. The ability of the cells which were contacted withthe candidate compounds is compared with the same cells which were notcontacted for 68772 activity.

[0073] The assays may simple test binding of a candidate compoundwherein adherence to the cells bearing the 68772 polypeptide is detectedby means of a label directly or indirectly associated with the candidatecompound or in an assay involving competition with a labeled competitor.Further, these assays may test whether the candidate compound results ina signal generated by activation of the 68772 polypeptide, usingdetection systems appropriate to the cells bearing the 68772polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed.

[0074] The 68772 cDNA, protein and antibodies to the protein may also beused to configure assays for detecting the effect of added compounds onthe production of 68772 mRNA and protein in cells. For example, an ELISAmay be constructed for measuring secreted or cell associated levels of68772 protein using monoclonal and polyclonal antibodies by standardmethods known in the art, and this can be used to discover agents whichmay inhibit or enhance the production of 68772 (also called antagonistor agonist, respectively) from suitably manipulated cells or tissues.Standard methods for conducting screening assays are well understood inthe art.

[0075] The 68772 protein may be used to identify membrane bound orsoluble receptors, if any, through standard receptor binding techniquesknown in the art. These include, but are not limited to, ligand bindingand crosslinking assays in which the 68772 is labeled with a radioactiveisotope (eg 125I), chemically modified (eg biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (cells, cell membranes, cellsupernatants, tissue extracts, bodily fluids). Other methods includebiophysical techniques such as surface plasmon resonance andspectroscopy. In addition to being used for purification and cloning ofthe receptor, these binding assays can be used to identify agonists andantagonists of 68772 which compete with the binding of 68772 to itsreceptors, if any. Standard methods for conducting screening assays arewell understood in the art.

[0076] Examples of potential 68772 polypeptide antagonists includeantibodies or, in some cases, oligonucleotides or proteins which areclosely related to the ligands, substrates, receptors, enzymes, etc., asthe case may be, of the 68772 polypeptide, e.g., a fragment of theligands, substrates, receptors, enzymes, etc.; or small molecules whichbind to the polypeptide of the present invention but do not elicit aresponse, so that the activity of the polypeptide is prevented.

[0077] Thus in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for 68772 polypeptides; or compounds whichdecrease or enhance the production of 68772 polypeptides, whichcomprises:

[0078] (a) a 68772 polypeptide, preferably that of SEQ ID NO:2;

[0079] (b) a recombinant cell expressing a 68772 polypeptide, preferablythat of SEQ ID NO:2;

[0080] (c) a cell membrane expressing a 68772 polypeptide; preferablythat of SEQ ID NO: 2; or

[0081] (d) antibody to a 68772 polypeptide, preferably that of SEQ IDNO: 2.

[0082] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0083] Prophylactic and Therapeutic Methods

[0084] This invention provides methods of treating abnormal conditionssuch as, proliferative diseases such as leukemias, solid tumor cancersand metastases; chronic inflammatory proliferative diseases such aspsoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, related to both an excess of and insufficient amounts of68772 polypeptide activity.

[0085] If the activity of 68772 polypeptide is in excess, severalapproaches are available. One approach comprises administering to asubject an inhibitor compound (antagonist) as hereinabove describedalong with a pharmaceutically acceptable carrier in an amount effectiveto inhibit the function of the 68772 polypeptide, such as, for example,by blocking the binding of ligands, substrates, enzymes, receptors,etc., or by inhibiting a second signal, and thereby alleviating theabnormal condition. In another approach, soluble forms of 68772polypeptides still capable of binding the ligand, substrate, enzymes,receptors, etc. in competition with endogenous 68772 polypeptide may beadministered. Typical embodiments of such competitors comprise fragmentsof the 68772 polypeptide.

[0086] In still another approach, expression of the gene encodingendogenous 68772 polypeptide can be inhibited using expression blockingtechniques. Known such techniques involve the use of antisensesequences, either internally generated or separately administered. See,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, FL (1988). Alternatively, oligonucleotides which formtriple helices with the gene can be supplied. See, for example, Lee etal., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al, Science (1991) 251:1360. These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

[0087] For treating abnormal conditions rated to an under-expression of68772 and its activity, several approaches are also available. Oneapproach comprises administering to a subject a therapeuticallyeffective amount of a compound which activates 68772 polypeptide, i.e.,an agonist as described above, in combination with a pharmaceuticallyacceptable carrier, to thereby alleviate the abnormal condition.Alternatively, gene therapy may be employed to effect the endogenousproduction of 68772 by the relevant cells in the subject. For example, apolynucleotide of the invention may be engineered for expression in areplication defective replication vector, as discussed above. Theretroviral expression construct may then be isolated and introduced intoa packaging cell transduced with a retroviral plasmid vector containingRNA encoding a polypeptide of the present invention such that thepackaging cell now produces infectious viral particles containing thegene of interest. These producer cells may be administered to a subjectfor engineering cells in vivo and expression of the polypeptide in vivo.For overview of gene therapy, see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics, T Strachan and A P Read, BIOSScientific Publishers Ltd (1996). Another approach is to administertherapeutic amount of 68772 polypeptides in combination with a suitablepharmaceutical carrier.

[0088] Formulation and Administration

[0089] Peptides, such as the soluble form of 68772 polypeptides, andagonists and antagonist peptides or small molecules, may be formulatedin combination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

[0090] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0091] Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

[0092] The dosage range required depends on the choice of peptide, theroute of administration, the nature of the formulation, the nature ofthe subject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thatadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

[0093] Polypeptides used in treatment can also be generated endogenouslyin the subject, in treatment modalities often referred to as “genetherapy” as described above. Thus, for example, cells from a subject maybe engineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES

[0094] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the ark exceptwhere otherwise described in detail. The examples illustrate, but do notlimit the invention.

Example 1

[0095] The full-length clone (68772) was identified through searches ofthe Human Genome Sciences database.

[0096] Northern blotting of multiple tissue human RNA blots performedusing 68772 (human pelota) as a probe, detected a message ofapproximately 1.9 kb in several cancer cell lines: HL-60, HelaS3, K-562,MOLT-4, Raji, SW480, A549, and G361. The message was also found in fetalliver, peripheral blood lymphocytes, and also weakly expressed in bonemarrow and thymus. No apparent message was detected in brain, spleen,appendix, lymph node, heart, placenta, lung, liver, skeletal muscle,kidney, or pancreas.

[0097] All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

1 2 1 1632 DNA human 1 cccgggcgct gcagtgttcc ccgagcctgt tagacgcagcgcgccgggag actgagagag 60 gaaaggatag aggaagtgct gccctaggct gcatgagtcgaagcaagcgt gtttccttcc 120 cgccaggcaa gtgcccttag aaaccgggcc ccgcccccttcctggcctgc attcccatcc 180 cctctcccgg ggcggaggtg aggacctcct tggttcctttggttctgtca gtgagcccct 240 tccttggcca tgaagctcgt gaggaagaac atcgagaaggacaatgcggg ccaggtgacc 300 ctggtccccg aggagcctga ggacatgtgg cacacttacaacctcgtgca ggtgggcgac 360 agcctgcgcg cctccaccat ccgcaaggta cagacagagtcctccacggg cagcgtgggc 420 agcaaccggg tccgcactac cctcactctc tgcgtggaggccatcgactt cgactctcaa 480 gcctgccagc tgcgggttaa ggggaccaac atccaagagaatgagtatgt caagatgggg 540 gcttaccaca ccatcgagct ggagcccaac cgccagttcaccctggccaa gaagcagtgg 600 gatagtgtgg tactggagcg catcgagcag gcctgtgacccagcctggag cgctgatgtg 660 gcggctgtgg tcatgcagga aggcctcgcc catatctgcttagtcactcc cagcatgacc 720 ctcactcggg ccaaggtgga ggtgaacatc cctaggaaaaggaaaggcaa ttgctctcag 780 catgaccggg ccttggagcg gttctatgaa caggtggtccaggctatcca gcgccacata 840 cactttgatg ttgtaaagtg catcctggtg gccagcccaggatttgtgag ggagcagttc 900 tgcgactaca tgtttcaaca agcagtgaag accgacaacaaactgctcct ggaaaaccgg 960 tccaaatttc ttcaggtaca tgcctcctcc ggacacaagtactccctgaa agaggccctt 1020 tgtgacccta ctgtggctag ccgcctttca gacactaaagctgctgggga agtcaaagcc 1080 ttggatgact tctataaaat gttacagcat gaaccggatcgagctttcta tggactcaag 1140 caggtggaga aggccaatga agccatggca attgacacattgctcatcag cgatgagctc 1200 ttcaggcatc aggatgtagc cacacggagc cggtatgtgaggctggtgga cagtgtgaaa 1260 gagaatgcag gcaccgctag gatattctct agtcttcacgtttctgggga acagctcagc 1320 cagttgactg gggtagctgc cattctccgc ttccctgttcccgaactttc tgaccaagag 1380 ggtgattcca gttctgaaga ggattaatga ttgaaacttaaaattgagac aatcttgtgt 1440 ttcctaaact gttacagtac atttctcagc atccttgtgacagaaagctg caagaagggc 1500 actttttgat tcatacaggg atttcttatg tctttggctacactagatat tttgtgattg 1560 gcaagacatg tatttaaaca ataaactaaa aggaaataatctccacgtac taccaaaaaa 1620 aaaaaaaaaa aa 1632 2 385 PRT human 2 Met LysLeu Val Arg Lys Asn Ile Glu Lys Asp Asn Ala Gly Gln Val 1 5 10 15 ThrLeu Val Pro Glu Glu Pro Glu Asp Met Trp His Thr Tyr Asn Leu 20 25 30 ValGln Val Gly Asp Ser Leu Arg Ala Ser Thr Ile Arg Lys Val Gln 35 40 45 ThrGlu Ser Ser Thr Gly Ser Val Gly Ser Asn Arg Val Arg Thr Thr 50 55 60 LeuThr Leu Cys Val Glu Ala Ile Asp Phe Asp Ser Gln Ala Cys Gln 65 70 75 80Leu Arg Val Lys Gly Thr Asn Ile Gln Glu Asn Glu Tyr Val Lys Met 85 90 95Gly Ala Tyr His Thr Ile Glu Leu Glu Pro Asn Arg Gln Phe Thr Leu 100 105110 Ala Lys Lys Gln Trp Asp Ser Val Val Leu Glu Arg Ile Glu Gln Ala 115120 125 Cys Asp Pro Ala Trp Ser Ala Asp Val Ala Ala Val Val Met Gln Glu130 135 140 Gly Leu Ala His Ile Cys Leu Val Thr Pro Ser Met Thr Leu ThrArg 145 150 155 160 Ala Lys Val Glu Val Asn Ile Pro Arg Lys Arg Lys GlyAsn Cys Ser 165 170 175 Gln His Asp Arg Ala Leu Glu Arg Phe Tyr Glu GlnVal Val Gln Ala 180 185 190 Ile Gln Arg His Ile His Phe Asp Val Val LysCys Ile Leu Val Ala 195 200 205 Ser Pro Gly Phe Val Arg Glu Gln Phe CysAsp Tyr Met Phe Gln Gln 210 215 220 Ala Val Lys Thr Asp Asn Lys Leu LeuLeu Glu Asn Arg Ser Lys Phe 225 230 235 240 Leu Gln Val His Ala Ser SerGly His Lys Tyr Ser Leu Lys Glu Ala 245 250 255 Leu Cys Asp Pro Thr ValAla Ser Arg Leu Ser Asp Thr Lys Ala Ala 260 265 270 Gly Glu Val Lys AlaLeu Asp Asp Phe Tyr Lys Met Leu Gln His Glu 275 280 285 Pro Asp Arg AlaPhe Tyr Gly Leu Lys Gln Val Glu Lys Ala Asn Glu 290 295 300 Ala Met AlaIle Asp Thr Leu Leu Ile Ser Asp Glu Leu Phe Arg His 305 310 315 320 GlnAsp Val Ala Thr Arg Ser Arg Tyr Val Arg Leu Val Asp Ser Val 325 330 335Lys Glu Asn Ala Gly Thr Ala Arg Ile Phe Ser Ser Leu His Val Ser 340 345350 Gly Glu Gln Leu Ser Gln Leu Thr Gly Val Ala Ala Ile Leu Arg Phe 355360 365 Pro Val Pro Glu Leu Ser Asp Gln Glu Gly Asp Ser Ser Ser Glu Glu370 375 380 Asp 385

What is claimed is:
 1. An isolated polynucleotide comprising anucleotide sequence that has at least 80% identity over its entirelength to a nucleotide sequence encoding the 68772 polypeptide of SEQ IDNO:2; or a nucleotide sequence complementary to said isolatedpolynucleotide.
 2. The polynucleotide of claim 1 wherein saidpolynucleotide comprises the nucleotide sequence contained in SEQ IDNO:1 encoding the 68772 polypeptide of SEQ ID NO2.
 3. The polynucleotideof claim 1 wherein said polynucleotide comprises a nucleotide sequencethat is at least 80% identical to that of SEQ ID NO: 1 over its entirelength.
 4. The polynucleotide of claim 3 which is polynucleotide of SEQID NO:
 1. 5. The polynucleotide of claim 1 which is DNA or RNA.
 6. A DNAor RNA molecule comprising an expression system, wherein said expressionsystem is capable of producing a 68772 polypeptide comprising an aminoacid sequence, which has at least 80% identity with the polypeptide ofSEQ ID NO:2 when said expression system is present in a compatible hostcell.
 7. A host cell comprising the expression system of claim
 6. 8. Aprocess for producing a 68772 polypeptide comprising culturing a host ofclaim 7 under conditions sufficient for the production of saidpolypeptide and recovering the polypeptide from the culture.
 9. Aprocess for producing a cell which produces a 68772 polypeptide thereofcomprising transforming or transfecting a host cell with the expressionsystem of claim 6 such that the host cell, under appropriate cultureconditions, produces a 68772 polypeptide.
 10. A 68772 polypeptidecomprising an amino acid sequence which is at least 80% identical to theamino acid sequence of SEQ ID NO:2 over its entire length.
 11. Thepolypeptide of claim 10 which comprises the amino acid sequence of SEQID NO:2.
 12. An antibody immunospecific for the 68772 polypeptide ofclaim
 10. 13. A method for the treatment of a subject in need ofenhanced activity or expression of 68772 polypeptide of claim 10comprising: (a) administering to the subject a therapeutically effectiveamount of an agonist to said polypeptide; and/or (b) providing to thesubject an isolated polynucleotide comprising a nucleotide sequence thathas at least 80% identity to a nucleotide sequence encoding the 68772polypeptide of SEQ ID NO:2 over its entire length; or a nucleotidesequence complementary to said nucleotide sequence in a form so as toeffect production of said polypeptide activity in vivo.
 14. A method forthe treatment of a subject having need to inhibit activity or expressionof 68772 polypeptide of claim 10 comprising: (a) administering to thesubject a therapeutically effective amount of an antagonist to saidpolypeptide; and/or (b) administering to the subject a nucleic acidmolecule that inhibits the expression of the nucleotide sequenceencoding said polypeptide; and/or (c) administering to the subject atherapeutically effective amount of a polypeptide that competes withsaid polypeptide for its ligand, substrate, or receptor.
 15. A processfor diagnosing a disease or a susceptibility to a disease in a subjectrelated to expression or activity of 68772 polypeptide of claim 10 in asubject comprising: (a) determining the presence or absence of amutation in the nucleotide sequence encoding said 68772 polypeptide inthe genome of said subject; and/or (b) analyzing for the presence oramount of the 68772 polypeptide expression in a sample derived from saidsubject.
 16. A method for identifying compounds which inhibit(antagonize) or agonize the 68772 polypeptide of claim 10 whichcomprises: (a) contacting a candidate compound with cells which expressthe 68772 polypepide (or cell membrane expressing 68772 polypeptide) orrespond to 68772 polypeptide; and (b) observing the binding, orstimulation or inhibition of a functional response; or comparing theability of the cells (or cell membrane) which were contacted with thecandidate compounds with the same cells which were not contacted for68772 polypeptide activity.
 17. An agonist identified by the method ofclaim
 16. 18. An antagonist identified by the method of claim
 16. 19. Anisolated 68772 polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence having at least80% identity to a nucleotide sequence encoding the 68772 polypeptideexpressed by the cDNA insert deposited at the ATCC with Deposit NumberATCC 98438; and (b) a nucleotide sequence complementary to thenucleotide sequence of (a).
 20. A recombinant host cell produced by amethod of claim 9 or a membrane thereof expressing a 68772 polypeptide.